Intracellular Redistribution of Left Ventricular Connexin 43 Contributes to the Remodeling of Electrical Properties of the Heart in Insulin-resistant Elderly Rats

Abstract

The correlation between long-QT and connexin 43 (Cx43) status and localization in elderly rats was determined to demonstrate a correlation between insulin resistance (I-R), ischemia-reperfusion, aging, and heart dysfunction. Male Wistar rats are grouped as 24-month-old rats (Aged-group), those with metabolic syndrome (8 months old; MetS-group), or controls (8 months old; Con-group). Both experimental groups have long-QT and low heart rate. Immunohistochemical imaging and quantification showed marked decreases in Cx43 staining of intercalated disc with less localizations in the Aged-group and MetS-group. The lateralization of Cx43 on longitudinal cell membrane was significantly high in the MetS-group than in the Con-group with no significant change in the Aged-group. Its significant cytoplasmic internalization was higher in the Aged-group than in the MetS-group. There were marked decreases in phospho-Cx43 (pCx43) staining of intercalated disc with less localizations in both groups than in the Con-group. Furthermore, lateralization of pCx43 was significantly low in the Aged-group and MetS-group, whereas there were no significant changes in the cytoplasmic internalization of both groups compared with the Con-group. Furthermore, the ratio of pCx43 to Cx43 was significantly small in both groups. We determined increases in RhoA and endothelin-1 in both groups, further supporting decreases in pCx43. Our data indicate the important role of I-R on long-QT in aging heart through alterations in both Cx43 protein level and localizations, leading to an abnormal spreading of ventricular repolarization in I-R heart:

An increase in intercellular crosstalk and electrotonic coupling between cardiomyocytes and nonmyocytes reshapes the electrical conduction in the metabolic heart characterized by short QT intervals in ECGs

“…Representative histological microphotographs associated with Cx43 are given in Figure 5A. Opposite to our previous study performed in the late‐stage MetS rats, 26 we, here, observed an unchanged Cx43 expression level in the heart the MetS rats compared to the CON group of rats CON (Figure 5C). This is followed by a marked increase in its intercalated discs (ID) localization as shown by high‐density Cx43 immunostaining in the samples from the MetS rat hearts, whereas its immunostaining along the longitudinal cell membrane (LCM) showing its lateralization was not different from the CON group rats with a less immunostaining level in the cytoplasm (less internalization of Cx43).…”

“…The left ventricles were isolated to perform routine histological procedures, as described previously 26 . Briefly, the tissue samples were fixed in phosphate‐buffered 10% formalin for 96 h and then dehydrated.…”

“…Western blot analysis was performed to determine the relative protein expression and the phosphorylation levels of Cx43, compared to a reference protein GAPDH in the heart tissues, as described previously 26 . Briefly, an equal amount of protein preparations was run on SDS‐polyacrylamide gels and blotted with specific antibodies for Cx43 (Santa Cruz, sc‐9059, 1:500), pCx43 (Abcam, ab194928,1:500), and reference protein GAPDH (Cell Signaling, D16H11, 1:5000) to detect their protein levels.…”

“…The left ventricular sections were analyzed as immunohistochemically as described previously. 26 Briefly, following the routine follow-up stages, the samples were incubated overnight at 4°C with specific primary antibodies (Na V 1.5; 1:200, D9J75, Cell Signaling, α-SMA; 1:100 A-5228, Sigma Aldrich, CD68; 1:100 sc-7084, Cx43; 1:100, sc-9059; pCx43; 1:100, ab194928 GJA1 phospho-S368, Abcam). As a negative control, nonimmune IgG (1:100; cat.…”

“…The left ventricles were isolated to perform routine histological procedures, as described previously. 26 Briefly, the tissue samples were fixed in phosphate-buffered 10% formalin for 96 h and then dehydrated. Following the routine follow-up stages, the samples were stained with Mallory Azan (MA) to detect fibrosis status (Carl Zeiss Oberkochen-Germany).…”